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Validated reverse phase high performance liquid Chromatography method for simultaneous estimation of Valsartan Potassium and Amlodipine Besylate in tablet dosage form

Nitesh Maheshwari*
Pranveer Singh Institute of Technology, Kanpur - 208005
Corresponding Author: Nitesh Maheshwari E-mail: niteshbit1@yahoo.co.in
Date of Submission: 08-05-2013 Date of Acceptance: 29-05-2013 Conflict of Interest: NIL Source of Support: NONE
Copyright: © 2013 Nitesh Maheshwari et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.
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Abstract

This work is concerned with application of simple, economical, precise, accurate and reproducible reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of Valsartan potassium (VP) and Amlodipine besylate (AB) on RP C-18 Column (Inertsil ODS-2, 150 x 4.6 mm) using Methanol: Water (62:38), pH adjusted to 3.0 with O-phosphoric acid as mobile phase at a flow rate of 1.4 ml/min and the detection wavelength was 230 nm. The retention time for VP and AB was found to be 6.0 and 3.5 min, respectively. Proposed method was validated for precision, accuracy, linearity range, robustness and ruggedness.

Keywords:

Valsartan potassium, Amlodipine besylate, Reverse Phase High Performance Liquid Chromatography, Inertsil, retention time, C-18 column.

Introduction

Amlodipine Besylate (AB) is chemically 3-Ethyl-5- methyl (±)-2-[(2-aminoethoxy) methyl]-4-(2- chlorophenyl)-1, 4-dihydro-6-methyl-3, 5- pyridinedicarboxylate, mono-benzenesulphonate, is used for treating hypertension and angina pectoris [1] in the form of the besylate salt, Amlodipine besylate. It is official in IP [2], USP [3], EP [4] and BP [5] .Various analytical methods have been reported for the assay of Amlodipine besylate [6] in pure form as well as in pharmaceutical formulations. They include high performance liquid chromatography[7]- [12], reversed phase high performance liquid chromatography[13]- [16] , high performance thin layer chromatography [17]- [20], gas chromatography[21], gas chromatography–mass spectrometry[22], liquid chromatography with tandem mass spectrometry[23] and fluorimetry[24], derivative spectroscopy[25]- [26] simultaneous multicomponent mode of analysis and difference spectrophotometry Valsartan is chemically (S)-N-(1-Oxopentyl)-N-[[2'- (1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl]methyl]-Lvaline, is an orally active specific angiotensin II receptor blocker effective in lowering blood pressure in hypertensive patients[27].Various Analytical methods have been reported for the assay of Valsartan in pure form as well as in pharmaceutical formulations includes HPLC[28]- [30], LC-MS[31]- [33], Protein precipitation[34], Capillary electrophoresis[35] and simultaneous UV spectrophotometric methods[36]- [37].
Amlodipine and Valsartan keep blood vessels from narrowing, which lowers blood pressure and improves blood flow. The combination of Amlodipine and Valsartan is used to treat high blood pressure (hypertension) [38]. This medication is usually given after others have been tried without successful treatment of hypertension [39] - [40].
Till now, no RP-HPLC study on simultaneous estimation of Amlodipine and Valsartan in tablet dosage form in pharmaceutical preparations has been found in literature survey. As no method is reported for Amlodipine and Valsartan in tablet formulation, the aim of the present study was to develop accurate, precise and selective reverse phase HPLC assay procedure for the analysis of Amlodipine and valsartan in its tablet dosage form.

Materials and Methods:

Chemicals and Reagents
Amlodipine Besylate and Valsartan are obtained as gift samples from Ranbaxy research Laboratories, Gurgaon and Hetero drugs, Hyderabad respectively. Methanol (HPLC grade), MilliQ water (HPLC grade), Potassium dihydrogen orthophosphate and orthophosphoric acid were of reagent grade. The Pharmaceutical preparation of combination of Amlodipine and Valsartan tablets that is going under product development phase at Ranbaxy research laboratories is available in the ratio of 10:160 mg as tablets.
Instrumentation
A Gradient HPLC system is used of Waters 2695 with PDA detector. The HPLC system was equipped with Empower software for data processing.
Selection of Chromatographic Condition
The mobile phase containing Methanol: water (68:32), pH adjusted to 3.0 with Orthophosphoric acid was found to resolve VP and AB. Orthophosphoric acid was used for pH adjustment of buffer. The mobile phase was filtered on a 0.45 micron membrane filter and then ultrasonicated for 30 min. The flow rate was set to 1.4mL min-1. Both drugs showed good absorbance at 238 nm, which was selected as wavelength for further analysis. All determinations were performed at constant column temperature (15 ± 2ºC).
In order to optimize the LC separation of AB and VP, initially, wavelength of 238nm was selected for the UV detection because at this wavelength there was maximum overlap of the spectra of Amlodipine and Valsartan. Nylon filter 0.45μm was selected for sample filtering as very good sample condition was observed against other filters. Retention of both the drugs was found dependent on pH of buffer. Amlodipine retention increases with increase in pH while Valsartan retention decreases with increase in pH. Both drugs were found sensitive to aqueous composition. A ten percent increase in aqueous composition resulted in 1.6 and 1.8 times increase in retention for amlodipine and Valsartan respectively. The buffer solution of pH3.0 and mobile phase composition of Methanol: water (62:38) was found most appropriate for separation of amlodipine and valsartan on Inertsil ODS-2 (150*4.6) mm, 5μm column. Flow rate of 1.4mL min-1 selected based on capacity factor and column efficiency. Amlodipine and Valsartan were well resolved in reasonable time of 8 minutes. The retention times were 3.5 min and 6.0 min, respectively. The resolution between amlodipine and valsartan was 14. The interference with the blank was within the limit of 1 percent. The peak purity of the peaks of Amlodipine and Valsartan was tested using PDA detector and were found to be pure.
Preparation of Stock Solutions
Standard stock solutions containing Valsartan Potassium (VP) and Amlodipine besylate (AB) were prepared by dissolving 25mg equivalent weight of Amlodipine and 160mg of Valsartan to a 250mL and 100mL volumetric flask respectively. 50mL of Methanol was added to each flask and sonicated for 20min to dissolve. Volume was made up to mark with mobile phase. 10mL of each solution was transferred to 100mL volumetric flask and the volume was made with mobile phase. Combined solution was filtered through 0.45 μm nylon filters to get stock solutions containing 160μgmL-1 of VP and 10μgmL-1 of AB respectively.
Sample Preparation
20 intact tablets were transferred to a 1000mL volumetric flask to which 100mL of Methanol was added and kept in ultrasonic bath for 10 minutes for complete dispersion of tablets. The volume was made up to mark with mobile phase. 5mL of the solution was transferred to 100mL volumetric flask and the volume was made with mobile phase and filtered through 0.45 μm nylon filter. The diluted solution was analyzed under optimized chromatographic conditions and chromatogram is depicted in figure no.1.
Method Validation [41]
The proposed HPLC method was validated as per ICH guidelines.
Specificity
The peak purity of VP and AB were assessed by comparing the retention time (TR) of standard VP and AB. Good correlation was obtained between the retention time of standard and sample of VP and AB. Peak purity is described in figure no. 2 and figure no. 3 respectively for amlodipine and valsartan peaks. Results are shown in Table 1.
Linearity and Range
Linearity was studied by preparing standard solutions at different concentration levels. The linearity range for VP and AB were found to be 112μgmL-1 to 208μgmL-1 and 07μgmL-1 to 13μgmL-1 respectively. The regression equation for VP and AB were found to be y = 28.30x + 63.62 and y = 28.78x + 98.30 with coefficient of correlation, (r) 0.99936 and 0.99914, respectively. Results are shown in Table 1.
Precision
Repeatability
Repeatability was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. Results are shown in Table 1.
Intermediate precision
Six independent sample solutions were prepared as per methodology and Intermediate precision was performed in duplicate on different HPLC system with different column on different date by different analyst but on same homogeneous sample batch. Overall %RSD was found to be less than 2%. Results are shown in Table 1.
Accuracy (Recovery studies)
To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard VP and AB were added to pre-analyzed samples and were subjected to the proposed HPLC method. Results of recovery studies are shown in Table 1.
Robustness of method

Results and Discussion

To develop a precise, accurate and suitable RPHPLC method for the simultaneous estimation of VP and AB, different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination. The results obtained by the validation of the combined formulation are summarized in Table 1. System suitability tests were carried out as per ICH guidelines are summarized in Table.2.

Conclusions

The proposed method is simple, sensitive and reproducible and hence the method can be used in routine for simultaneous determination of VP and AB in bulk as well as in pharmaceutical preparations. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The RSD for all parameters was found to be less than one, which indicates the validity of method and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative simultaneous estimation of VP and AB in multi component pharmaceutical preparation.

Acknowledgements:

The authors are thankful to the Principal Dr. Awanii Rai, Pranveer Singh Institute of Technology, Kanpur, Uttar Pradesh for providing necessary facilities and Ranbaxy research Lab.,Gurgaon and Hetero Drugs, Hyderabad for providing the gift sample of Amlodipine Besylate and Valsartan Potassium respectively. We are also thankful to Dr Swapana Mitra sir for his moral support and guidance. We are also very thankful to Mr. Vijay Lahkar for his honorable support throughout the analysis carried out.

Tables at a glance

Table icon Table icon
Table 1 Table 2

Figures at a glance

Figure 1 Figure 2 Figure 3
Figure 1 Figure 2 Figure 3

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